DNA-binding proteins as site-specific nucleases |
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Authors: | Clark Q. Pan Rail Landgraf David S. Sigman |
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Affiliation: | Molecular Biology Institute, UCLA, 405 Hilgard Avenue, Los Angeles, California 90024-1570, USA. |
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Abstract: | DNA-binding proteins can be converted into site-specific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor groove-proximal amino acid to a cysteine residue using site-directed mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10- phenanthroline-copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high-resolution nuclear magnetic resonance and X-ray crystal structures, and to locate binding sites of proteins within genomes. |
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